- Email: [email protected]
The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
遗 传 学 报 Acta Genetica Sinica, September
2006, 33 (9)：808–813
ISSN 0379-4172
The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep HUANG Zhi-Guo1,2, XIONG Li2, LIU Zhen-Shan1, QIAO Yong1, DAI Rong3, XIE Zhuang1, ①, LIU Shou-Ren1,3, SHI Guo-Qing3, LIU Guo-Qing1,3,① 1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 2. Liquor-making Biotechnology&Application Key Laboratory of Sichuan Province, Sichuan University of Science&Engineering, Zigong 643000, China; 3. Xinjiang Reclamation Science Institute, Shihezi 832001, China Abstract: Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01). Key words: sheep; ghrelin; abomasum; real-time PCR
Ghrelin, initially found in rat stomach tissue in 1999 by Kojima [1], is a brain-gut peptide containing 28 amino acids and exhibits growth hormone-releasing activities. Ghrelin was originally discovered as the endogenous ligand of growth hormone secretagogue receptor (GHSR) that could stimulate the release of growth hormone (GH) after binding to GHSR. However, it was soon established that ghrelin had a role in weight regulation because its administration increased food intake and caused fat and weight gain in rodents. Further studies found that ghrelin was involved in cardiac and gastrointestinal functions, cell proliferation, sleep, and so on. Human prepro-ghrelin displayed 82.9% homol-
ogy with that of rat and both consisted of 117 amino acids. The described ovine prepro-ghrelin cDNA (520 bp) codes for 116 amino acids, of which 27 amino acids from the 24th to the 50th residue from the N-terminus are mature ghrelin sequences [2]. Ghrelin was secreted primarily by stomach, and in situ hybridization indicated that ghrelin mRNA existed in the region from the neck to the base of the oxyntic gland [1]. Other tissues with ghrelin expression included hypothalamus, intestine, pituitary, liver, kidney, placenta, pancreas, testicle, and so on. There are many articles about the ghrelin gene in humans, rodents, and pigs, but in ruminants, ghrelin gene such as ovine ghrelin has rarely been reported.
Received: 2005-10-28; Accepted: 2005-12-07 This work was supported by Doctor Foundation of Xinjiang Construction Corps (No. 2003-02). ①
Corresponding author. E-mail: [email protected]; Tel: +86-25-8439 5046; Fax: +86-25-8439 5314.
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
In this article, male Kazak sheep and Xinjiang fine wool sheep, with different growth rates during the early growth period, were selected for investigation of the tissue distribution and developmental changes of ghrelin mRNA expression.
809
ment of body weight, animals were slaughtered for tissue sampling, which included hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and the longissimus dorsi muscle. The removed samples were snap-frozen in liquid nitrogen and then stored at −80℃ for total RNA analysis later.
1
Materials and Methods
1. 1
1. 2
Animals
Twenty-four male Kazak sheep and 30 male Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90, and 120), were selected from the Ziniquan Sheep Pasture in Shihezi City of Xinjiang Autonomous Region for the present study; however, there was no 120-day-old Kazak sheep. After measure-
Primer design
According to the published sequences of ovine ghrelin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, oligonucleotide primer sets for the two genes were designed using Primer premier 5.0 software and described in detail in Table 1. GAPDH was used as an internal standard for the determination of targeted mRNA levels.
Table 1 Parameters of gene-specific primers for ghrelin and GAPDH Target genes
GenBank accession number
ghrelin
AB060699
Primer sequence
Product size (bp)
Annealing temperature
205
58
379
58
(℃)
F: 5′-GGAAGTCAGGAGGAAGGTG-3′ R: 5′-GGGAGAACAGACAGGTGGT-3′ F: 5′-ACTTTGGCATCGTGGAGG-3′ GAPDH
AF030943 R: 5′-GAAGAGTGAGTGTCGCTGTTG-3′
were assessed by its optical density ratio at 260/280
2.5 mmol/L Spermidine), and 0.4 mmol/L each of dNTP. RNA sample, random primer, dNTP, and sterile H2O (final volume 10 µL) were first mixed in a 0.5 mL microcentrifuge tube and incubated at 70℃ for 5 min, and cooled on ice for 2 min. The rest of the reagents were then added into the reaction tube to a final volume of 25 µL and incubated at 37℃ for 1 h. The reaction was terminated by heating at 95℃ for 5 min and quickly cooled on ice. RT products were
nm (OD260/OD280=1.8−2.0) and by electrophoresis
stored at −20℃.
with ethidium-bromide staining.
1. 3. 3 Polymerase chain reaction (PCR) RT products (0.5 µL) were amplified in a 10 µL PCR reaction containing 1 U Taq DNA polymerase (TaKaRa, Inc. Dalian, China), 1 µL of 10× PCR buffer (100 mmol/L Tris-HCl pH 8.3, 500 mmol/L KCl), 0.25 mmol/L each of dNTP, 1.25 mmol/L MgCl2, and 0.5 µmol/L each of gene-specific primers. The following amplification conditions were used: one cycle of 1 min at 94℃ followed by 40 PCR
1. 3
Total RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
1. 3. 1
Total RNA extraction
Total RNA was extracted using the acid-guanidinium thiocyanate/phenol chloroform extraction method
[3]
.
The extracted RNA was dissolved in DEPC-treated water and the concentration, purity, and integrity
1. 3. 2 Reverse transcription (RT) Two micrograms of total RNA was used for reverse transcription in a final volume of 25 µL containing 200 U MMLV reverse transcriptase (Promega, Inc. Madison, USA), 20 U RNase inhibitor (Promega, Inc. Madison, USA), 1 µg of random primer, 5 µL of 5× RT buffer (250 mmol/L Tris-HCl pH 8.3, 50 mmol/L MgCl2, 250 mmol/L KCl, 50 mmol/L DTT,
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cycles of 30 s at 94℃, 30 s at the annealing temperature of the primers, 30 s at 72℃, and a final extension for 5 min at 72℃. Correct length of the products was confirmed on a 8% polyacrylamide gel, which was subsequently analyzed with a computer flatbed scanner after silver staining. 1. 4 Cloning and sequence analysis of the amplified fragments PCR products were excised after being confirmed by electrophoresis on a 1% agarose gel and purified by V-gene DNA Purification Kit (V-gene Biotechnology Ltd., Hangzhou, China) according to the manufacturer’s manual and then cloned into pMD18-T simple vector. Subsequently, the ligation products were transformed into JM109 cells. Positive clones based on blue-white selection were picked out for plasmid extraction by V-gene Kit (V-gene Biotechnology Ltd., Hangzhou, China) according to the manufacturer’s recommendation and then identified by PCR using gene-specific primers. Plasmids containing inserts of the right size were sequenced by Invitrogen Biotechnology Co., Ltd. (Shanghai, China).
Acta Genetica Sinica
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2006
The reactions were repeated twice for every sample. Plasmid DNA with the targeted DNA fragment was diluted to gradient concentrations, which were used to draw quantitative standard curves. 1. 6
Statistical analyses Data were described as x ± Sd and statistics
was analyzed using SPSS11.5 For Windows Software. Differences of body weight and gene expression level between ages in the same breed, and those at the same age between the two breeds were analyzed by one-way ANOVA and independent-samples t-test, respectively.
2 2. 1
Results Cumulative growth curves of sheep
Results showed that for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01) (Fig. 1).
1. 5 Real-time polymerase chain reaction (real-time PCR) The abundance of ghrelin mRNA was assessed by real-time reverse transcription polymerase chain reaction using a fluorescence temperature cycler (DNA Engine Opticon Real-time PCR Systems, MJ Research, Inc., Waltham, Massachusetts, USA). The final reaction volume was 20 µL containing 1 µL of the RT products, 1 U EX Taq HS DNA polymerase (TaKaRa Inc., Dalian, China), 4 µL of 5× PCR buffer, 0.3 mmol/L each of dNTP, 3.75 mmol/L MgCl2, 0.5 µmol/L each of primers, and 1 µL of 20× SYBR green. PCR conditions were as follows: one cycle of 1 min at 95℃; 45 PCR cycles of 10 s at 95℃, 10 s at the annealing temperature of the primers, 15 s at 72℃, plate-reading; this was followed by an extension of 10 min at 72℃; plate-reading every other 0.2℃ from 65℃ to 94℃ for drawing melting curves; and then the reaction was stopped with an extension of 5 min at 72℃.
Fig. 1 Cumulative growth curves of male Kazak sheep(HSK) and Xinjiang fine wool sheep(XJXM) Significant difference is denoted with letters (the capital for Xinjiang and the small for Kazak) and means without a common superscript indicate significant differences (P<0.05) between ages in the same breed. Double stars (**) indicate extreme differences (P<0.01) between breeds at the same age.
2. 2
RT-PCR of ghrelin and GAPDH genes
Total RNA from the abomasum of a Kazak sheep was used as an initial sample to amplify ghrelin and GAPDH genes by RT-PCR, which gave rise to a 205 bp and a 379 bp cDNA fragment, respectively (Fig. 2).
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
Fig. 2 RT-PCR of ghrelin and GAPDH mRNA in abomasums 1, 2: ghrelin; 3, 4: DNA marker pUC18; 5, 6: GAPDH.
2. 3 Sequence analysis of the amplified fragments The amplified ghrelin and GAPDH cDNA fragments were then cloned into pMD18-T simple vector. After ligation, the products were identified by PCR using gene-specific primers (Fig.3), with those plasmids containing inserts of the right size being sequenced. The sequences of the amplified fragments were aligned by DNAstar software with the corresponding reported sequences according to which the gene-specific primers were designed. The results showed that (1) there was 99.51% sequence identity between the amplified ghrelin gene cDNA fragment and the published ovine ghrelin gene sequence; (2) there was 100% sequence identity for the GAPDH gene. These results indicated that the amplified cDNA fragments of the two genes were gene-specific products.
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Fig. 3 PCR of ghrelin-pMD18-T and GAPDH-pMD18-T 1: ghrelin; 2,3: DNA marker pUC18; 4: GAPDH.
2. 4 Distribution of ghrelin mRNA expression in various tissues Two-day-old male Kazak sheep were used to investigate the distribution of ghrelin mRNA expression in various tissues including hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle by RT-PCR. It was found that ghrelin mRNA existed in all of the above tissues (Fig. 4). Further study using real-time PCR (Fig. 5) showed that ghrelin mRNA level was significantly higher in the abomasum than in other tissues (P<0.05) (Table 2).
Fig. 4 RT-PCR of ghrelin gene in sheep various tissues 1: pituitary; 2: hypothalami; 3: heart; 4: liver; 5: muscle; 6: duodenum; 7: rumen; 8: reticulum; 9: omasum; 10: abomasum; 11: DNA marker pUC18.
Fig. 5 The amplification, standard, and melting curves of ghrelin and GAPDH A, B, and C are the amplification, standard, and melting curves of ghrelin, respectively; D, E, and F are the amplification, standard, and melting curves of GAPDH, respectively.
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Table 2
Acta Genetica Sinica
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2006
Abundance of ghrelin mRNA in various tissues Tissues
Rumen
Heart
ghrelin/GAPDH ( x ± Sd )
0.103±0.04
Tissues
Muscle
ghrelin/GAPDH ( x ± Sd )
a
Liver
0.115±0.044
a
Hypothalamus a
0.263±0.048
0.415±0.05
a
Omasum
0.128±0.041
a
Pituitary
Reticulum
0.142±0.057
a
Duodenum ab
1.000±0.397
0.138±0.041a Abomasum
5.852±0.827
b
116.443±13.201c
Note: Significant difference is denoted by letters and means without a common superscript differ significantly between tissues (P<0.05).
2. 5
Developmental changes of ghrelin mRNA expression in sheep abomasum
Real-time PCR was used to quantify ghrelin mRNA levels in the abomasa of Kazak and Xinjiang fine wool sheep at different ages (Fig. 5). Results showed that the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90 ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01) (Fig.6).
in various sheep tissues, including the hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle, although it was expressed primarily in abomasum. The predominant expression of ghrelin mRNA in abomasum was consistent with results found in humans, rats, and so on. An in vitro study of rat showed that ghrelin specifically activated GHSR to stimulate GH release
[4]
and caused significant release of GH-releasing hormone (GHRH), but exerted no effect on the release of somatostatin (SS) [5]. Nakazato et al.[6] also found that intravenous administration of ghrelin to freely moving rats caused a dose-dependent increase in GH release. Furthermore, the stimulatory effect of ghrelin on GH-release was more intense than that of GHRH and hexarelin
[7]
. Fletcher et al.[8] found that ghrelin
was able to stimulate GH release, but not GH synthesis. Another study discovered that the effects of ghrelin on food intake increase were dose-dependent, even in GH-deficient rats [9], which suggested that the effects on food intake were independent of those on GH secretion. Chronic ghrelin administration has been shown to increase body fat content in rodents [10], which suggests that it may be related to adipogenesis Fig. 6 The developmental changes of abomasum ghrelin mRNA expression in male Kazak sheep(HSK) and Xinjiang fine wool sheep(XJXM) Significant difference is denoted by letters (the capital for Xinjiang and the small for HSK) and means without a common superscript differ significantly (P<0.05) between ages in the same breed; double stars (**) indicate extreme differences (P<0.01) between breeds of the same age.
3
Discussion It was found that ghrelin mRNA was expressed
and storage of energy. These data predict that ghrelin gene may play an important role in the process of food intake, weight regulation, and growth. The present research showed that abomasum ghrelin mRNA level increased gradually during early postnatal sheep development, a pattern that was similar to the changes observed in the cumulative growth curves. It was further found that from birth to day 90, ghrelin mRNA level in the abomasum of Kazak was significantly higher than that of Xinjiang, which was
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
in line with the fact that the body weight of Kazak was significantly higher (P<0.01) than that of Xinjiang during the same period. All these data suggested that ghrelin gene might be closely related to the growth and development of sheep. The present research established the basis for further studies on the effect of ghrelin gene on the growth and development of sheep and their regulatory mechanisms. References: [1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth hormone releasing acylated peptide from stomach. Nature, 1999, 402(6762) : 656-660. [2] http://www.ncbi.nlm.nih.gov/entrez/viewer.fegi?db= protein&val=30140885. [3] GAO Qin-Xue. Study on the characteristics of histology and molecular biology of intramuscular adipose in growing Erhualian pigs [Dissertation]. Nanjing Agricultural University, 2003. [4] Hayashida T, Murakami K, Mogi K, Nishihara M, Nakazato M, Mondal M S, Horii Y, Kojima M, Kangawa K, Murakami N. Ghrelin in domestic animals: distribution in stomach and its possible role. Domest Anim Endocrinol, 2001; 21(1) : 17-24. [5] Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshi-
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moto A, Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K. Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab, 2000, 85(12) : 4908-4911. [6] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature, 2001, 409(6817) : 194-198. [7] Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kangawa K. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun, 2000, 276(3) : 905-908. [8] Fletcher T P, Thomas G B, Clarke I J. Growth hormone-releasing hormone and somatostatin concentrations in the hypophysial portal blood of conscious sheep during the infusion of growth hormone-releasing peptide-6. Domest Anim Endocrinol, 1996, 13(3) : 251-258. [9] Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi T, Inoue G, Hosoda K, Kojima M, Kangawa K, Nakao K. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes, 2001, 50(2) : 227-232. [10] Tschöp M, Smiley D L, Heiman M L. Ghrelin induces adiposity in rodents. Nature, 2000, 407(6806) : 908-913.
绵羊 ghrelin 基因表达的组织分布和发育性变化 黄治国 1,2，熊 俐 2，刘振山 1，乔 永 1，代 蓉 3，谢 庄 1，刘守仁 1,3，石国庆 3，刘国庆 1,3 1. 南京农业大学动物科技学院，南京 210095； 2. 四川理工学院酿酒生物技术及应用四川省重点实验室，自贡 643000； 3. 新疆农垦科学院畜牧兽医研究所，石河子 832000 摘 要：选取2、30、60、90和120日龄的雄性哈萨克羊和新疆细毛羊各6只（无120日龄的哈萨克羊），测体重后屠宰，采下 丘脑、垂体、心脏、肝脏、瘤胃、网胃、瓣胃、皱胃、十二指肠、背最长肌，用RT-PCR和荧光实时定量PCR法检测ghrelin 基因表达的组织分布，及其在皱胃中的发育性变化。研究结果表明：（1）品种内各生长时期的体重差异显著（P<0.05）。 雄性哈萨克羊和新疆细毛羊的体重在2日龄时无显著差异（P>0.05），30～90日龄间，前者的体重极显著高于后者（P<0.01）； （2）所检测的各组织中都有 ghrelin mRNA 分布，但主要在皱胃中表达，其表达量远高于其他组织（P<0.05）；（3）两品 种绵羊皱胃 ghrelin 基因表达的发育性变化模式基本相似，都随着日龄的增加而呈上升趋势，其中雄性哈萨克羊的表达量 在2～60日龄间持续上升，60日龄后趋于水平；雄性新疆细毛羊的表达量在2～90日龄间持续上升，90日龄后趋于水平。研 究还发现雄性哈萨克羊皱胃 ghrelin 基因的表达量在2～90日龄间极显著高于新疆细毛羊（P<0.01）。 关键词：绵羊；ghrelin；皱胃；荧光实时定量 PCR 作者简介：黄治国（1978-），男，博士，研究方向：分子数量遗传学。E-mail: [email protected]
2006, 33 (9)：808–813
ISSN 0379-4172
The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep HUANG Zhi-Guo1,2, XIONG Li2, LIU Zhen-Shan1, QIAO Yong1, DAI Rong3, XIE Zhuang1, ①, LIU Shou-Ren1,3, SHI Guo-Qing3, LIU Guo-Qing1,3,① 1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 2. Liquor-making Biotechnology&Application Key Laboratory of Sichuan Province, Sichuan University of Science&Engineering, Zigong 643000, China; 3. Xinjiang Reclamation Science Institute, Shihezi 832001, China Abstract: Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01). Key words: sheep; ghrelin; abomasum; real-time PCR
Ghrelin, initially found in rat stomach tissue in 1999 by Kojima [1], is a brain-gut peptide containing 28 amino acids and exhibits growth hormone-releasing activities. Ghrelin was originally discovered as the endogenous ligand of growth hormone secretagogue receptor (GHSR) that could stimulate the release of growth hormone (GH) after binding to GHSR. However, it was soon established that ghrelin had a role in weight regulation because its administration increased food intake and caused fat and weight gain in rodents. Further studies found that ghrelin was involved in cardiac and gastrointestinal functions, cell proliferation, sleep, and so on. Human prepro-ghrelin displayed 82.9% homol-
ogy with that of rat and both consisted of 117 amino acids. The described ovine prepro-ghrelin cDNA (520 bp) codes for 116 amino acids, of which 27 amino acids from the 24th to the 50th residue from the N-terminus are mature ghrelin sequences [2]. Ghrelin was secreted primarily by stomach, and in situ hybridization indicated that ghrelin mRNA existed in the region from the neck to the base of the oxyntic gland [1]. Other tissues with ghrelin expression included hypothalamus, intestine, pituitary, liver, kidney, placenta, pancreas, testicle, and so on. There are many articles about the ghrelin gene in humans, rodents, and pigs, but in ruminants, ghrelin gene such as ovine ghrelin has rarely been reported.
Received: 2005-10-28; Accepted: 2005-12-07 This work was supported by Doctor Foundation of Xinjiang Construction Corps (No. 2003-02). ①
Corresponding author. E-mail: [email protected]; Tel: +86-25-8439 5046; Fax: +86-25-8439 5314.
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
In this article, male Kazak sheep and Xinjiang fine wool sheep, with different growth rates during the early growth period, were selected for investigation of the tissue distribution and developmental changes of ghrelin mRNA expression.
809
ment of body weight, animals were slaughtered for tissue sampling, which included hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and the longissimus dorsi muscle. The removed samples were snap-frozen in liquid nitrogen and then stored at −80℃ for total RNA analysis later.
1
Materials and Methods
1. 1
1. 2
Animals
Twenty-four male Kazak sheep and 30 male Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90, and 120), were selected from the Ziniquan Sheep Pasture in Shihezi City of Xinjiang Autonomous Region for the present study; however, there was no 120-day-old Kazak sheep. After measure-
Primer design
According to the published sequences of ovine ghrelin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, oligonucleotide primer sets for the two genes were designed using Primer premier 5.0 software and described in detail in Table 1. GAPDH was used as an internal standard for the determination of targeted mRNA levels.
Table 1 Parameters of gene-specific primers for ghrelin and GAPDH Target genes
GenBank accession number
ghrelin
AB060699
Primer sequence
Product size (bp)
Annealing temperature
205
58
379
58
(℃)
F: 5′-GGAAGTCAGGAGGAAGGTG-3′ R: 5′-GGGAGAACAGACAGGTGGT-3′ F: 5′-ACTTTGGCATCGTGGAGG-3′ GAPDH
AF030943 R: 5′-GAAGAGTGAGTGTCGCTGTTG-3′
were assessed by its optical density ratio at 260/280
2.5 mmol/L Spermidine), and 0.4 mmol/L each of dNTP. RNA sample, random primer, dNTP, and sterile H2O (final volume 10 µL) were first mixed in a 0.5 mL microcentrifuge tube and incubated at 70℃ for 5 min, and cooled on ice for 2 min. The rest of the reagents were then added into the reaction tube to a final volume of 25 µL and incubated at 37℃ for 1 h. The reaction was terminated by heating at 95℃ for 5 min and quickly cooled on ice. RT products were
nm (OD260/OD280=1.8−2.0) and by electrophoresis
stored at −20℃.
with ethidium-bromide staining.
1. 3. 3 Polymerase chain reaction (PCR) RT products (0.5 µL) were amplified in a 10 µL PCR reaction containing 1 U Taq DNA polymerase (TaKaRa, Inc. Dalian, China), 1 µL of 10× PCR buffer (100 mmol/L Tris-HCl pH 8.3, 500 mmol/L KCl), 0.25 mmol/L each of dNTP, 1.25 mmol/L MgCl2, and 0.5 µmol/L each of gene-specific primers. The following amplification conditions were used: one cycle of 1 min at 94℃ followed by 40 PCR
1. 3
Total RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
1. 3. 1
Total RNA extraction
Total RNA was extracted using the acid-guanidinium thiocyanate/phenol chloroform extraction method
[3]
.
The extracted RNA was dissolved in DEPC-treated water and the concentration, purity, and integrity
1. 3. 2 Reverse transcription (RT) Two micrograms of total RNA was used for reverse transcription in a final volume of 25 µL containing 200 U MMLV reverse transcriptase (Promega, Inc. Madison, USA), 20 U RNase inhibitor (Promega, Inc. Madison, USA), 1 µg of random primer, 5 µL of 5× RT buffer (250 mmol/L Tris-HCl pH 8.3, 50 mmol/L MgCl2, 250 mmol/L KCl, 50 mmol/L DTT,
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cycles of 30 s at 94℃, 30 s at the annealing temperature of the primers, 30 s at 72℃, and a final extension for 5 min at 72℃. Correct length of the products was confirmed on a 8% polyacrylamide gel, which was subsequently analyzed with a computer flatbed scanner after silver staining. 1. 4 Cloning and sequence analysis of the amplified fragments PCR products were excised after being confirmed by electrophoresis on a 1% agarose gel and purified by V-gene DNA Purification Kit (V-gene Biotechnology Ltd., Hangzhou, China) according to the manufacturer’s manual and then cloned into pMD18-T simple vector. Subsequently, the ligation products were transformed into JM109 cells. Positive clones based on blue-white selection were picked out for plasmid extraction by V-gene Kit (V-gene Biotechnology Ltd., Hangzhou, China) according to the manufacturer’s recommendation and then identified by PCR using gene-specific primers. Plasmids containing inserts of the right size were sequenced by Invitrogen Biotechnology Co., Ltd. (Shanghai, China).
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The reactions were repeated twice for every sample. Plasmid DNA with the targeted DNA fragment was diluted to gradient concentrations, which were used to draw quantitative standard curves. 1. 6
Statistical analyses Data were described as x ± Sd and statistics
was analyzed using SPSS11.5 For Windows Software. Differences of body weight and gene expression level between ages in the same breed, and those at the same age between the two breeds were analyzed by one-way ANOVA and independent-samples t-test, respectively.
2 2. 1
Results Cumulative growth curves of sheep
Results showed that for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01) (Fig. 1).
1. 5 Real-time polymerase chain reaction (real-time PCR) The abundance of ghrelin mRNA was assessed by real-time reverse transcription polymerase chain reaction using a fluorescence temperature cycler (DNA Engine Opticon Real-time PCR Systems, MJ Research, Inc., Waltham, Massachusetts, USA). The final reaction volume was 20 µL containing 1 µL of the RT products, 1 U EX Taq HS DNA polymerase (TaKaRa Inc., Dalian, China), 4 µL of 5× PCR buffer, 0.3 mmol/L each of dNTP, 3.75 mmol/L MgCl2, 0.5 µmol/L each of primers, and 1 µL of 20× SYBR green. PCR conditions were as follows: one cycle of 1 min at 95℃; 45 PCR cycles of 10 s at 95℃, 10 s at the annealing temperature of the primers, 15 s at 72℃, plate-reading; this was followed by an extension of 10 min at 72℃; plate-reading every other 0.2℃ from 65℃ to 94℃ for drawing melting curves; and then the reaction was stopped with an extension of 5 min at 72℃.
Fig. 1 Cumulative growth curves of male Kazak sheep(HSK) and Xinjiang fine wool sheep(XJXM) Significant difference is denoted with letters (the capital for Xinjiang and the small for Kazak) and means without a common superscript indicate significant differences (P<0.05) between ages in the same breed. Double stars (**) indicate extreme differences (P<0.01) between breeds at the same age.
2. 2
RT-PCR of ghrelin and GAPDH genes
Total RNA from the abomasum of a Kazak sheep was used as an initial sample to amplify ghrelin and GAPDH genes by RT-PCR, which gave rise to a 205 bp and a 379 bp cDNA fragment, respectively (Fig. 2).
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
Fig. 2 RT-PCR of ghrelin and GAPDH mRNA in abomasums 1, 2: ghrelin; 3, 4: DNA marker pUC18; 5, 6: GAPDH.
2. 3 Sequence analysis of the amplified fragments The amplified ghrelin and GAPDH cDNA fragments were then cloned into pMD18-T simple vector. After ligation, the products were identified by PCR using gene-specific primers (Fig.3), with those plasmids containing inserts of the right size being sequenced. The sequences of the amplified fragments were aligned by DNAstar software with the corresponding reported sequences according to which the gene-specific primers were designed. The results showed that (1) there was 99.51% sequence identity between the amplified ghrelin gene cDNA fragment and the published ovine ghrelin gene sequence; (2) there was 100% sequence identity for the GAPDH gene. These results indicated that the amplified cDNA fragments of the two genes were gene-specific products.
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Fig. 3 PCR of ghrelin-pMD18-T and GAPDH-pMD18-T 1: ghrelin; 2,3: DNA marker pUC18; 4: GAPDH.
2. 4 Distribution of ghrelin mRNA expression in various tissues Two-day-old male Kazak sheep were used to investigate the distribution of ghrelin mRNA expression in various tissues including hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle by RT-PCR. It was found that ghrelin mRNA existed in all of the above tissues (Fig. 4). Further study using real-time PCR (Fig. 5) showed that ghrelin mRNA level was significantly higher in the abomasum than in other tissues (P<0.05) (Table 2).
Fig. 4 RT-PCR of ghrelin gene in sheep various tissues 1: pituitary; 2: hypothalami; 3: heart; 4: liver; 5: muscle; 6: duodenum; 7: rumen; 8: reticulum; 9: omasum; 10: abomasum; 11: DNA marker pUC18.
Fig. 5 The amplification, standard, and melting curves of ghrelin and GAPDH A, B, and C are the amplification, standard, and melting curves of ghrelin, respectively; D, E, and F are the amplification, standard, and melting curves of GAPDH, respectively.
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Table 2
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Abundance of ghrelin mRNA in various tissues Tissues
Rumen
Heart
ghrelin/GAPDH ( x ± Sd )
0.103±0.04
Tissues
Muscle
ghrelin/GAPDH ( x ± Sd )
a
Liver
0.115±0.044
a
Hypothalamus a
0.263±0.048
0.415±0.05
a
Omasum
0.128±0.041
a
Pituitary
Reticulum
0.142±0.057
a
Duodenum ab
1.000±0.397
0.138±0.041a Abomasum
5.852±0.827
b
116.443±13.201c
Note: Significant difference is denoted by letters and means without a common superscript differ significantly between tissues (P<0.05).
2. 5
Developmental changes of ghrelin mRNA expression in sheep abomasum
Real-time PCR was used to quantify ghrelin mRNA levels in the abomasa of Kazak and Xinjiang fine wool sheep at different ages (Fig. 5). Results showed that the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90 ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01) (Fig.6).
in various sheep tissues, including the hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle, although it was expressed primarily in abomasum. The predominant expression of ghrelin mRNA in abomasum was consistent with results found in humans, rats, and so on. An in vitro study of rat showed that ghrelin specifically activated GHSR to stimulate GH release
[4]
and caused significant release of GH-releasing hormone (GHRH), but exerted no effect on the release of somatostatin (SS) [5]. Nakazato et al.[6] also found that intravenous administration of ghrelin to freely moving rats caused a dose-dependent increase in GH release. Furthermore, the stimulatory effect of ghrelin on GH-release was more intense than that of GHRH and hexarelin
[7]
. Fletcher et al.[8] found that ghrelin
was able to stimulate GH release, but not GH synthesis. Another study discovered that the effects of ghrelin on food intake increase were dose-dependent, even in GH-deficient rats [9], which suggested that the effects on food intake were independent of those on GH secretion. Chronic ghrelin administration has been shown to increase body fat content in rodents [10], which suggests that it may be related to adipogenesis Fig. 6 The developmental changes of abomasum ghrelin mRNA expression in male Kazak sheep(HSK) and Xinjiang fine wool sheep(XJXM) Significant difference is denoted by letters (the capital for Xinjiang and the small for HSK) and means without a common superscript differ significantly (P<0.05) between ages in the same breed; double stars (**) indicate extreme differences (P<0.01) between breeds of the same age.
3
Discussion It was found that ghrelin mRNA was expressed
and storage of energy. These data predict that ghrelin gene may play an important role in the process of food intake, weight regulation, and growth. The present research showed that abomasum ghrelin mRNA level increased gradually during early postnatal sheep development, a pattern that was similar to the changes observed in the cumulative growth curves. It was further found that from birth to day 90, ghrelin mRNA level in the abomasum of Kazak was significantly higher than that of Xinjiang, which was
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
in line with the fact that the body weight of Kazak was significantly higher (P<0.01) than that of Xinjiang during the same period. All these data suggested that ghrelin gene might be closely related to the growth and development of sheep. The present research established the basis for further studies on the effect of ghrelin gene on the growth and development of sheep and their regulatory mechanisms. References: [1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth hormone releasing acylated peptide from stomach. Nature, 1999, 402(6762) : 656-660. [2] http://www.ncbi.nlm.nih.gov/entrez/viewer.fegi?db= protein&val=30140885. [3] GAO Qin-Xue. Study on the characteristics of histology and molecular biology of intramuscular adipose in growing Erhualian pigs [Dissertation]. Nanjing Agricultural University, 2003. [4] Hayashida T, Murakami K, Mogi K, Nishihara M, Nakazato M, Mondal M S, Horii Y, Kojima M, Kangawa K, Murakami N. Ghrelin in domestic animals: distribution in stomach and its possible role. Domest Anim Endocrinol, 2001; 21(1) : 17-24. [5] Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshi-
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moto A, Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K. Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab, 2000, 85(12) : 4908-4911. [6] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature, 2001, 409(6817) : 194-198. [7] Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kangawa K. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun, 2000, 276(3) : 905-908. [8] Fletcher T P, Thomas G B, Clarke I J. Growth hormone-releasing hormone and somatostatin concentrations in the hypophysial portal blood of conscious sheep during the infusion of growth hormone-releasing peptide-6. Domest Anim Endocrinol, 1996, 13(3) : 251-258. [9] Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi T, Inoue G, Hosoda K, Kojima M, Kangawa K, Nakao K. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes, 2001, 50(2) : 227-232. [10] Tschöp M, Smiley D L, Heiman M L. Ghrelin induces adiposity in rodents. Nature, 2000, 407(6806) : 908-913.
绵羊 ghrelin 基因表达的组织分布和发育性变化 黄治国 1,2，熊 俐 2，刘振山 1，乔 永 1，代 蓉 3，谢 庄 1，刘守仁 1,3，石国庆 3，刘国庆 1,3 1. 南京农业大学动物科技学院，南京 210095； 2. 四川理工学院酿酒生物技术及应用四川省重点实验室，自贡 643000； 3. 新疆农垦科学院畜牧兽医研究所，石河子 832000 摘 要：选取2、30、60、90和120日龄的雄性哈萨克羊和新疆细毛羊各6只（无120日龄的哈萨克羊），测体重后屠宰，采下 丘脑、垂体、心脏、肝脏、瘤胃、网胃、瓣胃、皱胃、十二指肠、背最长肌，用RT-PCR和荧光实时定量PCR法检测ghrelin 基因表达的组织分布，及其在皱胃中的发育性变化。研究结果表明：（1）品种内各生长时期的体重差异显著（P<0.05）。 雄性哈萨克羊和新疆细毛羊的体重在2日龄时无显著差异（P>0.05），30～90日龄间，前者的体重极显著高于后者（P<0.01）； （2）所检测的各组织中都有 ghrelin mRNA 分布，但主要在皱胃中表达，其表达量远高于其他组织（P<0.05）；（3）两品 种绵羊皱胃 ghrelin 基因表达的发育性变化模式基本相似，都随着日龄的增加而呈上升趋势，其中雄性哈萨克羊的表达量 在2～60日龄间持续上升，60日龄后趋于水平；雄性新疆细毛羊的表达量在2～90日龄间持续上升，90日龄后趋于水平。研 究还发现雄性哈萨克羊皱胃 ghrelin 基因的表达量在2～90日龄间极显著高于新疆细毛羊（P<0.01）。 关键词：绵羊；ghrelin；皱胃；荧光实时定量 PCR 作者简介：黄治国（1978-），男，博士，研究方向：分子数量遗传学。E-mail: [email protected]